ATP-dependent proteases play critical roles in the post-translational regulation of the amounts and activities of important cellular proteins and in the removal of damaged and denatured proteins from the cell. Our research has focussed on the biochemical characterization of the ATP- dependent Clp and Lon proteases from E. coli and on the discovery of new ATP-dependent proteases. Site-directed mutagenesis of the two ATPase sites in ClpA demonstrated that the active form of ClpA is a hexamer and that interactions between the subunits affect the ATPase and protease- activating activity of ClpA. The domain I site was shown to be involved in assembly of the ClpA hexamer, and interaction with ClpP and the domain II site was needed for activation of the proteolytic activity ClpP against large proteins. Model peptide substrates have been synthesized and shown to be cleaved more rapidly than any previous substrates. These peptides interacted tightly with ClpA and activated its ATPase activity. The site on ClpA occupied by the peptides defines the allosteric site for proteins on the enzyme, and occupancy of this site appears to be necessary to open the active site of ClpP for large polypeptides and to accelerate the catalytic cleavage of peptide bonds. Using partially inactivated ClpP, it was demonstrated that processive cleavage of polypeptides and proteins required the array of active sites present in the dodecameric form of ClpP. Processive cleavage of model peptides was observed when non- hydrolyzable analogs of ATP were used to activate the enzyme, indicating that ATP hydrolysis does not play a direct role in processivity. Studies done in collaboration with Dr. Susan Gottesman have demonstrated that another ATPase from E. coli, ClpX, is evolutionarily related to ClpA and that this protein functions in vivo with ClpP as an essential component of a proteolytic system that degrades the highly unstable lambda O protein. Sequence and biochemical data now indicate the presence in E. coli of four ATPases, Clps A, B, X and Y and suggest that the energy-dependent proteolytic systems of even this simple organism are highly complex. In studies done in collaboration with Drs. Michael Gottesman and Nan Wang, it has been found that a close homolog of the ATP-dependent Lon protease occurs in human mitochondria. Further biochemical and immunochemical studies are underway to identify the properties and function of this protease in human cells.